Bovine vibriosis vaccine and method of administering same



United States Patent 3,329,573 BOVINE VIBRIOSIS VACCINE AND METHOD OF ADMINISTERING SAME Alvin B. Hoerlein, Fort Collins, Colo., assignor to Colorado State University Research Foundation, Fort Collins, Colo., a non-profit corporation of Colorado No Drawing. Filed Aug. 26, 1963, Ser. No. 304,649 9 Claims. (Cl. 167-78) This invention relates to a vaccine together with the method of administering same that is capable of increasing the resistance to bovine vibriosis, a disease caused by the bacterium Vibrio fetus.

Vibriosis is a venereal infection of cattle caused by Vibrio fetus that produces temporary infertility. The disease has been known since about 1918 and many investigators have since determined its causative agent. Until recently the disease appeared to be confined to dairy herds and small beef herds where it could be controlled rather easily by using artificial insemination techniques. If the semen itself contains no Vibrio fetus, either because it was taken from non-infected bulls or because it Was treated with antibiotics to kill any of these and other organisms present before use, then spread of the disease can be effectively prevented. Unfortunately, however, these relatively simple control measures become utterly ineffective in range herds. For instance, in a large range herd, artificial insemination becomes a practical impossibility.

Relatively recently, bovine vibriosis has been detected in substantial numbers of range cattle and the control thereof has become a factor of major economic importance to cattlemen who rely heavily on minimum increases in their herd to break even. Until now, however, science has found no practical way to control this disease by artificial means in a range herd and, in fact, the prior art suggests, and even teaches, that such means will not be effective. It is of utmost importance, therefore, to understand the nature of this disease and how it differs from the common internal infectious diseases where the bacteria invade body tissues and a classical immune response could be expected from vaccine stimulation. It is the discovery that bovine vibriosis which can be characterized as an external infectious disease can, in fact, be controlled by a vaccine injected into the tissues that constitutes the novelty of the present invention, especially when the teaching of the prior art as developed by highly skilled investigators concludes that the standard immunological techniques will not work.

An understanding of the completely unexpected nature of this discovery can, perhaps, best be realized by comparing bovine vibriosis with ovine vibriosis, the latter being one of many internal infectious diseases which can be prevented by the more or less standard immunization techniques. First, let us look at the similarities between bovine and ovine forms of the disease.

Both diseases involve Vibrio fetus and are genital in nature. In each, reproduction is unsuccessful, in bovine vibriosis through invasion of the embryo itself, in ovine vibriosis due to destruction of the maternal and fetal placental tissues resulting in death of the approximately full-term fetus. Here, however, the similarity ends.

In the case of ovine vibriosis, the bacteria gain entrance into the body by ingestion, migrate through the body in the blood stream and eventually invade the uterus thereby causing an abortion of the advanced fetus to take place by reason of the placental tissue destruction. It is this fact of invasion of the maternal tissue by the bacteria which makes it reasonable to expect that a classical immune response would result from parenteral introduction of Vibrio fetus antigens by artificial means. In other -words, the fact that the bacteria are known to move through the ewes blood stream and eventually locate in her uterus is, in and of itself, ample reason to expect that immunity could result in an ewe injected with Vibrio fetus antigens when challenged with ovine vibriosis. Thus, the pathogenesis of ovine vibriosis is similar to most other infectious diseases.

Bovine vibriosis, on the other hand, has no counterpart in man or other animals, but rather, possesses a unique pathogenesis among the infectious diseases. It is known, for example, as an external disease because the bacteria never invade the maternal body tissue but invade only the fetal embryo. When a cow has been bred by an infected bull, Vibrio fetus is deposited in the uterine cavity and begins to propagate in the copious estrual secretions of the uterus. These bacteria do not pass beyond the uterine epithelium or even produce any inflammatory reactions in the uterus; however, they do invade the fetal embryo causing it to die while still contained within the uterine lumen usually before any attachment to the uterus. The failure to find any clear histopathologic changes in the endometrium of infected cattle provides ample support for the foregoing pathogenesis.

One might contend that because the transient presence of agglutination reactions in vaginal mucus and serum occur following infection with Vibrio fetus, that this fact is related to classical antibody production in the usual immunological sense; but, no such relationship has been shown and, in fact, is very questionable since false agglutination reactions often occur when serum and cervicovaginal mucus is used that has been taken from cattle that have never been infected with Vibrio fetus. Furthermore, even if the aforementioned reactions were, in fact, due to the presence of true agglutinins, little, if any, weight could be given such a finding for the simple reason that it is well known from studies of other diseases that there is no correlation between immunity and the presence of agglutinins. Finally, there is ample evidence to support the finding that these agglutination reactions have nothing whatsoever to do with resistance to reinfection. Noteworthy among this evidence is the fact that hyperimmune serum does not prevent vibriosis in cattle.

About the only fact which would lend credence to a suspicion that the customary immune reactions would occur when parenteral introductions of Vibrio fetus antigens were made by artificial means is that recovered animals are resistant to reinfection with Vibrio fetus. Why this resistance to reinfection occurs is not presently known but one plausible theory holds that it is what may be denominated a clinical resistance to infertility and the abortion-producing effects of embryonal infection by Vibrio fetus.

Be that as it may, whatever suspicions that may have existed at one time concerning the normal immune response to injections of Vibrio fetus antigens when challenged with the virulent bacteria were, once and for all, dispelled when a recognized authority in the field failed to produce immunity to bovine vibriosis following injections of a modified live culture of Vibrio fetus. These unsuccessful experiments have been accepted by most, if not all, of the recognized authorities on bovine vibriosis as conclusive evidence of the fact that introduction of Vibrio fetus antigens will not evoke a classical immune response to a challenge by the virulent organism due to the external nature of the disease as distinguished from that of ovine vibriosis and many other infectious diseases that follow the normal pattern. Evidence of the acceptance of the previously-mentioned unsuccessful experiments as final proof of the conclusions therein reached is found by examining the reports of world-renowned experts on vibriosis which make no mention of the entire subject of induced resistance to infection.

Contrary to this accepted belief, it has now been found in accordance with the teaching of the present invention that induced resistance to infection by Vibrio fetus can, in fact, be accomplished in cattle with either dead or live organisms.

It is, therefore, the principal object of the present invention to provide the first known successful procedure for preventing a disease that involves introduction of the protective organism into the body of a host whose tissues are never invaded by the infectious organism.

A second object is to provide a unique vaccine effective to increase the resistance of cattle to infection ordinarily resulting from the venereal introduction of Vibrio fetus.

Another object of the invention herein claimed is the provision of a novel method for preventing bovine vibriosis in cattle which comprises parenteral administration of a vaccine prepared from Vibrio fetus of either bovine or ovine origin.

Still another objective is the provision of a product that has proven effective in the control of bovine vibriosis when made up from live Vibrio fetus cultures or dead ones killed by various chemicals as well as heat and alternate freezing and thawing.

An additional object of the invention herein claimed is the provision of a bovine vibriosis vaccine that exhibits surprising effectiveness in various adjuvants.

Further objects are the provision of a means and method for controlling bovine vibriosis which is effective for use in range herds, versatile, safe in that no untoward side effects have been noted, relatively simple to prepare and use, and seemingly independent of the particular strain or strains of Vibrio fetus employed.

Other objects will be in part apparent and in part pointed out specifically hereinafter in connection with the detailed descriptive material which follows.

The cultures of Vibrio fetus used for vaccine production originated from two strains isolated from near-fullmorphology characteristic of young Vibrio fetus. Those colonies meeting these criteria were then immediately transferred to Bactio thiol semi-solid agar (0.4% agar) in screw-capped culture tubes to exclude oxygen and incubated at 37 C. for three days. Those cultures having typical sub-surface growth were again examined microscopically for purity and desirable cell morphology. If satisfactory, the mass of bacterial growth and the semisolid agar in the area in which growth occurred was transferred to Bactio thiol agar (2.0% agar) in Roux bottles which were rubber stoppered to exclude oxygen. After incubation for two days at 37 C., the bacterial growth was harvested by the various methods appropriate to the particular vaccine being prepared.

The above technique was the one used for the production of mass growth of Vibrio fetus for all of the vaccines prepared, however, other methods known in the art will, no doubt, produce comparable results and there is no intention of limiting the scope of the invention herein described to the foregoing method of propagating the cultures.

The initial tests were performed using a live culture vaccine. The growth of Vibrz'o fetus, after 2 days incubation in the Roux bottles, was first examined for purity and characteristic cell morphology. After suspending the bacteria in Albimi Brucella broth, the suspension was adjusted so that a 1/100 dilution had an OD. of 0.32 (550 me). This suspension was injected without delay into experimental heifers, subcutaneously, in 10 ml. doses for Experiment I. This injection was repeated 24 days later.

This initial experiment (Experiment I) demonstrated that heifers injected subcutaneously with living Vibrio fetus could be effectively rendered resistant to vibriosis as evidenced by the results given in Table I which follows:

TABLE I.SUMMARY OF RESULTS FOR EXPERIMENT I SHOWING RE SIS'IANCE TO INFECTION WITH VIBRIO FETUS FOLLOWING SUBCU- TANEOUS INJECTION OF LIVING VIBRIO FETUS *Very highly significant (P: .001).

term aborted bovine feti from two infected herds. These cultures were pooled and serially passed through experimental heifers by intracervical introduction and reisolation from cervical mucus. Between passages the cultures were preserved by rapid freezing in 10% glycerinated Albimi brucella broth or by lyophilizing in sterile pig serum to prevent deterioration of the desired properties which stimulate the production of resistance to infection. Just before preparing the vaccines the cultures were serially passaged in experimental heifers to enhance both virulence and antigenicity before propagation of the bacteria for vaccine production. In addition, an ovine strain of Vibrio fetus isolated from an aborted ovine fetus was also tested. While only these specific strains of Vibrio fetus were employed, there is no reason to suspect that they are unique or that other strains would not function equally well if properly handled.

A minimum of artificial propagation of the bacteria was desirable to prevent deterioration of those properties which evoke resistance to vibriosis; therefore, cervical mucus from passage heifers was inoculated onto blood agar plates and incubated in an atmosphere of approximately 10% CO and 90% N at 37 C. for 3 days. The bacterial growth of typical isolated colonies was then examined microscopically to determine purity and the The next experiment (Experiment II) was devised to find a practicable vaccine since the previous experiment had shown that it was possible to induce resistance to vibriosis by artificial means. The first step was an investigation of adjuvant effect.

Suspensions of formalin-killed Vibrio fetus were emulsified with a mineral-oil-lanolin adjuvant and a sodium alginate adjuvant to determine the relative enhancing effect of these substances. After injection of these adjuvant bacterins into experimental heifers, it was found that the production of serum agglutinins was similar for both of these adjuvants and lower than that produced with live cultures. The mineral oil base adjuvant was chosen for further investigation since it produced a persistent granuloma at the injection site which suggested a more prolonged antigenic stimulation of the suspended bacteria.

The mineral-oil-lanolin adjuvant that was selected was prepared for the above killed-vaccines by adding 18 parts of refined lanolin to 82 parts of light mineral oil with heat. It was sterilized by steam under pressure at a temperature of 121 C. for 30 minutes. Each of the hereinafter described killed suspensions of Vibrio fetus was emulsified with an equal volume of the adjuvant by repeated expulsion through hypodermic needle using a syringe, until a water-in-oil emulsion was formed.

This emulsion was a smooth, creamy material which flowed through a 16 gauge needle slowly even after warming the body temperature, and was stable for at least 30 days.

The mineral-oil-lanolin adjuvant could be replaced with other water-non-miscible substances. Other non-toxic stabilizers could, no doubt, be used in place of lanolin in mineral oil base adjuvants. If such non-toxic stabilizers lowered the viscosity without loss of effect they would even add to the convenience of use. The actual emulsification of larger amounts of vaccine could be accomplished more easily through the use of high speed mixers of the Waring Blendor type, in a colloid mill of the Eppenbach type, or in a disperator of the Premier type. While enhancing bacteria such as Mycobacterium spp. could probably be added to the adjuvant, their use would not seem warranted in view of the excellent results obtained without them. i

It was also necessary to determine whether Vibrio fetus could be killed without destroying the particular substance or substances responsible for evoking the resistance to infection evidenced by injection of living organisms. To determine whether this could be accomplished by any means and also to determine the anatomical location in the bacterial cell (flagellar or somatic), and the general chemical nature of the substance or substances stimulating resistance to infection, several methods of killing the causative bacteria were utilized.

In the preparation of a formalin-killed vaccine, the two-day growth of Vibrio fetus in Roux bottles described previously was removed after examination for purity and example, the alternate freezing and thawing above-described, enable certain portions of the organism having no antigenic effect to be discarded in favor of other portions which possess the desired immunological properties. Therefore, the term Vibrio fetus vaccine" as used herein is intended to connotate that product which increases the resistance to bovine vibriosis irrespective of whether said vaccine is produced from the whole bacterium or only that portion thereof possessing the necessary antigens to evoke an immune response.

All of the foregoing methods of killing Vibrio fetus for vaccine production proved effective, however, this is not to imply that other methods well known to the art might not produce comparable results. Thus, while several different procedures for killing Vibrio fetus to be used in the vaccine were tried and proven workable, they are by no means intended to be an exhaustive study of the several possible killing methods that could have been used. 1

In the second experiment (Experiment II) suspensions of killed Vibrio fetus were emulsified with the mineraloil-lanolin adjuvant. The resulting adjuvant vaccines were subsequently injected subcutaneously into experimental heifers in 5 ml. amounts to approximate the number of bacteria contained in the live culture vaccine. A second similar injection of vaccine was made 18 days later, ex-

TABLE II.-SUMMARY OF RESULTS OF EXPERIMENT II SHOWING RE- SISTANCE TO INFECTION WITH VIBRIO FETUS FOLLOWING SUBCU- TANEOUS INJECTION OF VARIOUS VIBRIO FETUS VACCINES Treatment Percent Breedings No. of Treatment before breeding at first Percent pregnant er heifers (vaccine) breeding pregnant from first pregnancy (challenge) breeding None Semen only- 90 70 1. 6 do Semen plus 10 0 39. 0

V. fetus. Living V. fetus* do 100 80 1.2 Formalin-killed V. fetus. 100 50 1.8 Dilute formalin-killed 100 1. 8

V. fetus. Phenol-killed V. fetus do 89 67 1.9 10 Heabkilled V. fetus do 80 3.0 10 V. fetus killed by freezing do 90 60 1. 8

and thawing.

*Replicate of vaccine used in Experiment I. cell morphology by suspension in 0.15% formolized sa- 50 line solution (0.15% formaldehyde in 0.9% NaCl, pH 7.0). The resulting suspension was adjusted so that a 1/200 dilution had an O.D. of 0.30 (550 mg). The dilute formalin-killed vaccine was diluted 1/20.

A phenol-killed vaccine was also prepared using the two-day growth of Vibrio fetus harvested from the Roux bottle cultures by suspension in a solution of 1.0% phenol in 0.9% NaCl. The resulting suspension of bacteria was adjusted so that a 1/200 dilution had an O.D. of 0.30 (550 m A heat killed-vaccine was produced from the two-day growth of Vibrio fetus in the Roux bottles which was harvested in 0.9% NaCl solution and the resulting suspension adjusted so that a 1/200 dilution had an O.D. of 0.29 (550 mu). This bacterial suspension was then heated in a water bath at 65 C. for 30 minutes to kill the bacteria.

Finally, a freeze-killed vaccine was prepared by taking the two-day growth of Vibrio fetus already mentioned and harvesting same from the Roux bottle cultures with Albimi Brucella broth. The resulting suspension was standardized so that a l/200 dilution had an O.D. of 0.31 (550 m Seven slow freezing and thawing cycles were necessary to kill all of the bacteria.

It is well known that some techniques for killing organisms to be used in vaccine production such as, for

While the different bacterins varied in their ability to stimulate resistance to infection, all of them demonstrated practicable usefulness in the control of vibriosis.

The particular chemical component or components of the bacterial cell responsible for the action desired is apparently somatic and not associated with flagellar substance, since phenol and heat did not destroy activity. The constituents of bacterial cells disintegrated by means of freezing and thawing were also still active in stimulation'of resistance to vibriosis. The differences between the vaccinated and unvaccinated experimental groups were all highly significant (P= .005). The vaccine groups were not significantly different from the negative control group which was not infected with vibriosis. The virulence of the challenge cultures is evidenced by the poor breeding results in the challenge controls in which only 10% were pregnant after four breedings.

From the foregoing, it will be apparent that the vaccine of the present invention comprises the first known means for immunizing cattle against bovine vibriosis in direct contradiction to the teaching of the prior art as established by internationally known skilled investigators of the disease. Furthermore, the pathogenesis of the disease is such that parenteral administration of a vaccine would not be expected .to produce the desired protection against Vibrio fetus in cattle due to the purely external nature thereof.

Thus the vaccine itself becomes novel due to the teaching of the prior art that a bovine vibriosis vaccine would be ineffective and, in addition, the method of administration thereof also becomes novel when the fact is recognized that the Vibrio fetus never invades the tissues of the cow, yet, it is the cow rather than the fetus which is inoculated. In other words, knowing the pathogenesis of bovine vibriosis, one might expect the vaccine to be effective if administered directly to the host whose tissues are invaded by the Vibrio fetus organism but not otherwise. Obviously, inoculation of the fetal embryo is a practical impossi bility for many reasons even if this situs for the vaccine did, in fact, prove effective. Also, the latter approach ignores the fact that the cow would already be infected at the time the fetus became available for treatment.

Having thus described in detail the novel features of the bovine vibriosis vaccine of the present invention together with its method of administration, it will be seen that the several worthwhile objectives for which it was developed have been achieved. While the foregoing descriptive material is, of necessity, limited to the results achieved when certain specific strains of Vibrio fetus were employed in formation of the vaccines and the vaccines themselves employed only a limited number of adjuvants as well as chemical and mechanical procedures for killing same, it is my intention that the invention herein claimed not be confined to those specific techniques and formulations hereinabove described because others will, of course, occur to those skilled in the art within the broad teaching hereof. Hence, it is my intention that the scope of protection afforded hereby shall be limited only insofar as said limitations are expressly set forth in the appended claims.

What is claimed is:

1. The vibriosis bacterin which comprises: virulent Vibrio fetus having insufficient inherent immunogenicity to invoke the desired immune response that has been modified by serial susceptible animal passage until its natural virulence has increased to a level that renders same highly immunogenic followed by killing.

2. The adjuvant vibriosis bacterin composition which comprises: the modified Vibrio fetus bacterin produced by the process of claim 1 suspended in an adjuvant selected from those adapted to produce prolonged antigenic stimulation of said suspended bacterin.

3. The adjuvant vibriosis bacterin composition as set forth in claim 2 in which: the adjuvant is oil-based.

4. The adjuvant vibriosis bacterin composition as set forth in claim 2 in which: the bacterin is diluted with water, and in which a water-immiscible adjuvant is mixed with the aqueous bacterin solution in sufficient quantities to form an emulsified suspension of said bacterin.

5. The adjuvant vibriosis bacterin composition as set forth in claim 2 in which: the adjuvant is selected from the group consisting of sodium alginate and mineral oil.

6. The adjuvant vibriosis bacterin composition as set forth in claim 2 which includes lanolin as a non-toxic stabilizer.

7. The method of increasing the resistance in cattle to infectious bovine vibriosis which comprises: injection into susceptible female cattle prior to conception of the vibriosis bacterin modified in accordance with the process of claim 1.

8. The method of increasing the resistance in cattle to infectious bovine vibriosis which comprises: injection into susceptible female cattle prior to conception of an adjuvant vibriosis bacterin composition including a vibriosis bacterin modified in accordance with the process of claim 1 suspended in an adjuvant adapted to produce prolonged antigenic stimulation of said suspended bacterin.

9. The method of increasing the resistance in cattle to infectious bovine vibriosis which comprises: subcutaneous injection into susceptible female cattle prior to conception of an aqueous suspension of a vibriosis bacterin modified in accordance with the process of claim 1 blended together with an oil-based adjuvant in sulficient quantities to form an oil-in-water emulsified suspension.

References Cited Miller et al.: American Journal of Veterinary Research, vol. 22, No. 86, pp. 43-46, January 1961.

Osborne: American Veterinary Medical Association Proceedings, 1952, pp. 112117.

Plastridge: Advances in Veterinary Research, vol. 2, pp. 370-371, 1955.

Plastridge et al.: Journal of the American Veterinary Medical Association, vol. 118, pp. 367-375.

Ristic et al.: American Journal of Veterinary Research, vol. 14, pp. 399-444, 1953.

Storis: Agricultural Experimental Station Bulletin No. 281, pp. 44 -47, 1951.

Te Punga: The New Zealand Veterinary Journal, vol. 10, No. 4, pp. 8991, 1962.

Wagner: Cornell Vet., vol. 5, pp. 441-450, 1961.

LEWIS GOTTS, Primary Examiner.

ELBERT L. ROBERTS, Examiner.

R. L. HUPF, Assistant Examiner. 

1. THE VIRIOSIS BACTERIN WHICH COMPRISES: VIRULENT VIBRIO FETUS HAVING INSUFFICIENT INHERENT IMMUNOGENICITY TO INVOKE THE DESIRED IMMUNE RESPONSE THAT HAS BEEN MODIFIED BY SERIAL SUSCEPTIBLE ANIMAL PASSAGE UNTIL ITS NATURAL VIRULENCE HAS INCREASED TO A LEVEL THAT RENDERS SAME HIGHLY IMMUNOGENIC FOLLOWED BY KILLING. 